The genetic diversity and relationships among plants belonging to the subfamily Sedoideae (Crassulaceae), some of which are indigenous to Korea or introduced from other countries, were determined using chloroplast (cp) nucleotide sequence analysis. To analyze genetic diversity and variation among 19 plants including species belonging to Sedum, Hylotelephium, and Phedimus, the tRNA-Leucine gene (trnL [UAA]) and adjoining spacer in chloroplast DNA (cpDNA) were sequenced and compared across species. Species were divided into two main groups based on the cpDNA sequence comparison. The generated phylogeny indicated that many native Sedum species had diverged from S. album. Members of the Phedimus and Hylotelephium species, and several Sedum species analyzed here, clustered distinctively in different groups. Using cpDNA sequence analysis, we successfully discriminated Sedoideae plants cultivated in Korea from each other, even at the intraspecific level, and the results were reflective of the morphological and biogeographical characteristics. These findings could be useful for classifying samples for proper naming, choosing breeding materials for new cultivars, or identifying species for conservation of horticultural crop resources.